Direct Measurement of Glycosaminoglycan Intermolecular Interactions via High-Resolution Force Spectroscopy

Abstract

Intermolecular repulsion forces between negatively charged glycosaminoglycan (CS-GAG) macromolecules are a major determinant of cartilage biomechanical properties. It is thought that the electrostatic component of the total intermolecular interaction is responsible for 50-75% of the equilibrium elastic modulus of cartilage in compression, while other forces (e.g., steric, hydration, van der Waals, etc.) may also play a role. To investigate these forces, radiolabeled CS-GAG polymer chains, with a fully extended contour length of 35 nm, were chemically end-grafted to a planar surface to form model biomimetic polyelectrolyte "brush" layers whose environment (e.g., ionic strength, pH) was varied to mimic physiological conditions. The total intersurface force (enN) between the CS-GAG brushes and chemically modified probe tips (SO 3 -and OH) was measured as a function of tip-substrate separation distance in aqueous solution using the technique of high-resolution force spectroscopy (HRFS). These experiments showed long-range, nonlinear, purely repulsive forces that decreased in magnitude and range with increasing ionic strength and decreasing pH. To estimate the contribution of the electrostatic component to the total intersurface force, the data were compared to a theoretical model of electrical double layer repulsion based on the Poisson-Boltzmann formulation. The CS-GAG brush layer was approximated as either a flat surface charge density or a smoothed volume of known fixed charge density and the probe tip was modeled as a smooth hemisphere of constant surface charge density. Modeling the CS-GAG brush as a volume charge yielded theoretical fits much closer to the experimental data and is a good first step toward deconvolution of the force components.

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…The authors could show that the Hamaker constant is dominated by the underlying gold support (for a calculation of the Hamaker constant of SAMs on metal supports as a function of the thickness of layers see also [347]). The same result has been found by Seog et al [348] by measuring the forces between glycosaminoglycan on gold and by Vezenov et al [349].…”

Synthesis and Single Molecule Force Spectroscopy of Graft Copolymers of Poly(2-hydroxyethyl methacrylate-g-ethylene glycol)

“…A full description of this instrument, its limits of force and displacement detection in fluids, procedures for spring constant calibration and conversion of raw data, details of measurement errors, and description of typical force vs distance curves including the mechanical instabilities of the cantilever, are given in our previous work. 41,42 Force (nN) vs distance (nm) curves were measured at room temperature using Thermomicroscopes microfabricated V-shaped Si 3N4 cantilevers with the square-pyramidal probe tip. The cantilevers were with the following dimensions: cantilever length of 320 µm, cantilever width of 22 µm, cantilever thickness of 0.6 µm, force constant of 0.01 N/m, and resonant frequency of 7 kHz.…”

Nanoscale Conformation and Compressibility of Cartilage Aggrecan Using Microcontact Printing and Atomic Force Microscopy

“…canol, HS(CH 2 ) 11 OH (Aldrich), in ethanol on a freshly cleaned 1 cm × 1 cm gold-coated substrate 10 for 30 s using a hexagonal patterned poly(dimethylsiloxane) (PDMS) stamp (hexagon side length ) 15 µm and spacing ) 15 µm) followed by incubation in 50 µL of 1 mg/mL thiol-terminated aggrecan solution for 48 h. Hence, the aggrecan was located within the hexagons and the OH-SAM outside the hexagons. OH-functionalized AFM gold-coated probe tips (Digital Instruments, k ∼ 0.06 N/m, tip radius ∼50 nm) were prepared by immersion in 4 mM 11-mercaptoundecanol for 48 h to minimize electrostatic interactions between the probe tip and aggrecan layer.…”

“…The carboxyl and sulfate groups on the CS-GAG are ionized at all bath pH and IS of Figure 3. 10 The electrostatic interaction distance in monovalent electrolyte solutions is determined by Illustration of AFM contact mode imaging on aggrecan and hydroxyl-terminated SAM patterned surface (Rtip ∼ 50 nm, aggrecan molecules spaced ∼20 nm apart, contour length ∼400 nm, side chain (mostly GAG) contour length ∼40 nm, side chains spaced ∼2-4 nm apart). permittivity (6.92 × 10 -10 C/(N m 2 )), F the Faraday constant (96 500 C/mol), R the universal gas constant, T the absolute temperature (298 K), and C 0 the bath IS).…”